Inhibitory Effective Perturbations of Cilobradine (DK-AH269), A Blocker of HCN Channels, on the Amplitude and Gating of Both Hyperpolarization-Activated Cation and Delayed-Rectifier Potassium Currents

Abstract

Cilobradine (CIL, DK-AH269), an inhibitor of hyperpolarization-activated cation current (Ih), has been observed to possess pro-arrhythmic properties. Whether and how CIL is capable of perturbing different types of membrane ionic currents existing in electrically excitable cells, however, is incompletely understood. In this study, we intended to examine possible modifications by it or other structurally similar compounds of ionic currents in pituitary tumor (GH3) cells and in heart-derived H9c2 cells. The standard whole-cell voltage-clamp technique was performed to examine the effect of CIL on ionic currents. GH3-cell exposure to CIL suppressed the density of hyperpolarization-evoked Ih in a concentration-dependent manner with an effective IC50 of 3.38 μM. Apart from its increase in the activation time constant of Ih during long-lasting hyperpolarization, the presence of CIL (3 μM) distinctly shifted the steady-state activation curve of Ih triggered by a 2-s conditioning pulse to a hyperpolarizing direction by 10 mV. As the impedance-frequency relation of Ih was studied, its presence raised the impedance magnitude at the resonance frequency induced by chirp voltage. CIL also suppressed delayed-rectifier K+ current (IK(DR)) followed by the accelerated inactivation time course of this current, with effective IC50 (measured at late IK(DR)) or KD value of 3.54 or 3.77 μM, respectively. As the CIL concentration increased 1 to 3 μM, the inactivation curve of IK(DR) elicited by 1- or 10-s conditioning pulses was shifted to a hyperpolarizing potential by approximately 10 mV, and the recovery of IK(DR) inactivation during its presence was prolonged. The peak Na+ current (INa) during brief depolarization was resistant to being sensitive to the presence of CIL, yet to be either decreased by subsequent addition of A-803467 or enhanced by that of tefluthrin. In cardiac H9c2 cells, unlike the CIL effect, the addition of either ivabradine or zatebradine mildly led to a lowering in IK(DR) amplitude with no conceivable change in the inactivation time course of the current. Taken together, the compound like CIL, which was tailored to block hyperpolarization-activated cation (HCN) channels effectively, was also capable of altering the amplitude and gating of IK(DR), thereby influencing the functional activities of electrically excitable cells, such as GH3 cells.

Effects of Sesamin, the Major Furofuran Lignan of Sesame Oil, on the Amplitude and Gating of Voltage-Gated Na+ and K+ Currents

“…Cells were exposed to Ca 2+ -free Tyrode's solution containing 1 μM TTX and the pipette was filled with K + -containing solution. As the hyperpolarizing command pulse from −40 to −110 mV with a duration of 2 sec was delivered, Ih with a slowly activating property was robustly evoked, as observed previously [16,38,39]. As illustrated in Figure 8, 1 min of exposure to SSM at a concentration of 10 μM was unable to modify the amplitude or gating (i.e., activation or deactivation kinetics) of Ih in response to a 2-sec hyperpolarizing pulse from −40 to −110 mV.…”

“…However, in the continued presence of SSM (10 μM), the subsequent application of cilobradine at a concentration of 3 or 10 μM was highly effective at inhibiting the I h amplitude in combination with a measurable slowing in the activation time course of the current. Cilobradine has recently been reported to decrease I h amplitude, as well as to alter activation kinetics present in different types of excitable cells [ 39 ]. As such, distinguishable from its effect on I Na or different types of K + currents demonstrated above, the addition of SSM failed to alter the amplitude and kinetics of I h identified in GH 3 cells.…”

Effective Activation by Kynurenic Acid and Its Aminoalkylated Derivatives on M-Type K+ Current

“…However, in the continued presence of 30 μM KYNA, further addition of 10 μM cilobradine was able to decrease I h amplitude to 41 ± 11 pA ( n = 8, p < 0.05). Cilobradine was previously reported to block I h [ 43 ]. Mean I-V relationship of I h amplitude obtained in the absence and presence of KYNA or KYNA plus 10 μM cilobradine is illustrated in Figure 8 B.…”

Involvement of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 3 in Oxytocin Neuronal Activity in Lactating Rats With Pup Deprivation

“…Then, we blocked HCN3 by using DK-AH269 ( Neitz et al., 2011 ; Lu et al., 2020 ) and then tested effects of PGE 2 on the electrical activity of oxytocin neurons ( Figure 9A ). DK-AH269 (10 μmol/L) significantly reduced the firing rate (5.9 ± 0.5 Hz, n = 10 in DK-AH269 vs. 11.1 ± 0.4 Hz, n = 10 before DK-AH269, p = .047 by Dunnett’s T3 test) and blocked PGE 2 -evoked increase in the firing rate (4.0 ± 0.6 Hz, n = 9 in DK-AH269 + PGE 2 , p = .775 to DK-AH269 by Dunnett’s T3 test; Figure 9Ac).…”